Antiviral compositions

ABSTRACT

The invention relates to antiviral compositions of matter containing as active ingredient hypericin or pseudohypericin. The novel compositions are of special use in the treatment of, and alleviation of symptoms of various diseases caused by viruses such as vesicular stomatitis, influenza, herpes simplex, HSV-1 and HSV-2. The mode of activity seems to be by inhibition of RNA synthesis and interfering with virus replication. The pharmaceutical compositions can be applied by a variety of routes. Topical applications have proved to be effective against a variety of viral afflictions.

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions of matter whichcontain as active ingredient hypericin or pseudohypericin. Thecompositions are effective in inhibiting viral replication and thusbring about the gradual destruction of the virus. The compositions areof value in systemic as well as topical application. They can be usedeffectively against vesicular stomatitis virus, influenza virus, herpessimplex HSV-1, HSV-2 and others. The active ingredient can be isolatedfrom the perennial plant Hypericum triquetrifolium Turra (or Hypericumcrispum L.).

SUMMARY OF THE INVENTION

There are provided antiviral pharmaceutical compositions which containas active ingredient hypericin or pseudohypericin. The compositions areeffective in interfering with the development and replication of virusesin mammals, including humans, inducing their destruction, without anyappreciable toxicity to the mammal.

BACKGROUND OF THE INVENTION

Only few compounds are known to possess antiviral activity. One of thebest is interferon, which is currently in experimental therapeutic usein humans. The production and purification of human interferon is fairlytedious and the available quantities are limited. However, much is knowntoday about the mechanism of action of this antiviral protein which actsby inducing an antiviral state in cells (Antiviral Drugs and Interferon:The molecular Basis of their Activity. Ed Yechiel Becker 1984, p. 357,Martinus Nijhoff Publishing Boston). The active ingredient, hypericinand pseudohypericin can be isolated and purified. The results of ourinvestigation led to the isolation from the plant Hypericumtriquetrifolium Turra, also known as Hypericum crispum L., of twocompounds identified chemically as hypericin (1) and pseudohypericin(2). These compounds have been isolated among others in a pure form andcharacterized using NMR techniques. Their spectra showed all thecharacteristic signals specific for their polycyclic aromaticstructures. ##STR1## These two compounds showed an unequivocal andstrong antiviral activity against vesicular stomatitis virus (VSV),influenza virus, herpes simplex HSV-1 and HSV-2 and others. Theirtoxicity was found to be low in tissue cultures and in mammals. Testsperformed on the incorporation of radioactive ³ H-uridine into RNA bythe virus-indicated that the activity of these two compounds involves aninhibition of RNA synthesis, interfering with the replication of thevirus, resulting in its destruction.

In order to display their activity, the compounds have to be in directcontact with the virus. The estimation of the antiviral activity isbased on protection of cells by the compounds from destruction by thevirus, previously put in contact with low concentrations of thecompounds tested.

In view of this behaviour the compounds are appropriate for topicalapplication in localized infections.

The compounds have been described earlier by Brockmann et al. (H.Brockmann and W. Sanne. Zur Kenntnis des Hypericins undPseudo-hypericins. Chem. Ber. 90, 2480, (1957)); H. Brockmann, U.Franssen, D. Spitzner and H. Augustiniak. Zur Isolierung undKonstitution des Pseudohypericins, Tetrahedron Letters, 1991, (1974))who have also provided the appropriate NMR signals for each of the twocompounds (1) and (2) (H. Brockmann and D. Spitzner. Die Konstitutiondes pseudohypericins, Tetrahedron Letters 37, (1975)). A synthesis forhypericin has also been reported (H. Brockmann, F. Kluge and H.Muxfeldt. Totalsynthese des Hypericins, Chem. Ber. 90, 2302 (1957)).Although species of the genus Hypericum (St. John's wort) have beenreported to display an antiviral activity (N.A. Derbentseva et al.,Effect of tannins from Hypericum perforatum on influenza virusMikrobiol. Zh. (Kiev) 34, 768 (1972) C.A. 78 67532d; Mishenkova et al.,Antiviral properties of St. John's wort and preparation produced fromit. Tr. S'ezda Mikrobiol. Ukr. 4th. 222 (1975), C.A. 85 187161Y), thetwo compounds hypericin (1) and pseudohypericin (2) have never beenreported or recorded to have antiviral activity.

Hypericin (1) is also described in the Merck Index (Eighth edition, O.G. Stecher Ed., 1968, p 558) as appearing to have a tonic andtranquilizing action on the human organism. It has been used in medicineas antidepressant (Daniel K. Further communication on the photodynamicsubstance hypericin. Hippokrates 20, 526 (1949); C.A. 46 9721/e (1952)).

CHEMICAL PREPARATION

The herb of the whole plant harvested at its flowering time was dried at55° C., cut and milled, and then extracted with acetone. 1 kg. of thematerial was put in a soxhlet and extracted for several hours (5-10 hr)until the extracting solvent was colorless. The solution assumed a redfluorescent color. The solvent was evaporated under reduced pressure tocomplete dryness of the residue (95 g). This residue was thenfractionated on a chromatographic column, packed with silica gel 60(0.06-0.20 mm). The chromatography applied was the dry process, whereby25 g of the residue dissolved in acetone are added to an equal amount ofsilica gel 60 and evaporated on a rotavapor with swirling untilhomogeneous and dry. The mixture was then placed at the top of thecolumn, (1/Kg) and eluted first with CH₂ Cl₂ until the solvent reachedthe bottom of the column, followed by a solvent mixture consisting ofCH₂ Cl₂ -acetone-MeOH 75:15:10 parts. When the red color became weaker,the concentration of the CH₂ Cl₂ was reduced so as to form a solventmixture consisting or 55:15:10 parts respectively. The fractionscollected were of about 250 ml each; they were monitored on thin layerchromatoplates observing the R_(f) value of the two main red fluorescentspots under ultraviolet light. The developing solvent mixture was asabove. The chromatography was completed in about 2 days. Furtherpurification and separation of the two main components was reachedthrough flash chromatography using silica gel 60 (mesh 0.04-0.06) withslight pressure and again using the dry approach with the same solventcombination. The two main components of interest were identified ashypericin R_(f) 0.45, yield 0.19 g and pseudohypericin R_(f) 0.35, yield0.73 g. NMR spectra identical with those reported in the literature(Brockmann (1975) ibid.).

BIO-ASSAYS

The antiviral tests were performed using the radioactive assay based onthe incorporation of radioactive ³ H-uridine into viral RNA in infectedcells. The cell cultures used in these tests were grown on Eagle'smedium and they originated from human fibroblast FS11 culturesestablished in our laboratory (J. Weissenbach, M. Zeevi, T. Landau andM. Revel. Identification of the translation products of human fibroblastinterferon mRNA in reticulocyte lysates. Eur. J. Biochem., 98 1-8(1979)). The virus used for these tests was vesicular stomatitis virus(VSV) proliferating on the cell cultures. In this test the antiviralactivity is expressed as reduction in incorporation of labelledmaterial, namely inhibition of viral replication. A simpler and morerapid method taking into consideration the toxicity of the testedcompounds to the cells was the cytophathic effect (CPE) monitored in thecells under the microscope. This system enables to follow the effect ofthe antiviral compounds on the cells for a number of days. It wascarried out using microtiter plates having 96 wells (8×12), and serialdilutions of the tested compounds were added in decreasing concentrationin order to determine the activity against their toxicity at the lowestconcentration. A relatively short incubation time (about 1 hr) followinga contact between the virus and the tested dilution of the compound wasallowed before adding to the cells in the wells, and then the cytopathiceffect was recorded at time intervals. An additional assay system usedwas the inhibition of plaque formation by virus in a monolayer culture.Here, absence of plaque formation indicates inhibition of viralreplication. This procedure was used in the case of influenza and herpessimplex. For the latter the following procedure was followed: serialdilutions of the hypericin and pseudohypericin solutions were preparedin PBS (phosphate buffered Saline). A constant amount of virus was addedto each dilution, and the mixtures were incubated for several hours at4° C., the mixtures were then used to infect monolayers of Vero cells in60 mm dishes, in the plaque assay, as follows:

1. Adsorption of virus (in the mixture), 0.2 ml/plate, 1 hr 37° C.

2. Addition of overlay: 2% FCS (Fetal calf serum) in methyl cellulose oragarose. Incubation 3 days in a humidified CO₂ incubator (the virusmixture was not removed after the 1 hr adsorption). 3. Counting plaquesafter staining.

RESULTS Vesicular stomatitis virus assays

Using the CPE method, the crude extract of the plant H. crispum showedactivity at concentrations of 325 γ/ml. A first purification led to amixture active at 85 γ/ml. At this stage the toxicity to the cells inthe culture was highly reduced. Following the sequence ofchromatographies described above the two compounds hypericin andpseudohypericin were obtained showing activity at a level of 5 γ/ml, seefor example charts 1 and 2. Chart 1 indicates the delay in destructionof VSV infected cells at a concentration of a few gamma per ml ofhypericin. At the optimum concentration of 9 γ/ml, the cells remainedviable for time periods up to 140 hrs. Chart 2 shows that at the sameconcentrations the cells in contact with the products alone (withoutvirus), remained viable throughout the same period of incubation.

For the radioactive bioassay the compound tested, pseudohypericin, wasput into the assay system following a previous contact of the virus withdifferent concentrations of the substance, and the mixture then added tothe cell cultures. The results are plotted in charts 3 and 4. It can beseen that the decrease in percent ³ H-uridine incorporation accountingfor inhibition of the RNA synthesis is observed not only on the VSVinfected cells, but also on the cells alone used as control. A definitedose response with the different dilutions was reached, and a nicerepetition of the results could be observed with two virus dilutions50×10⁶ p.f.u./well and 100×10⁶ p.f.u./well (p.f.u.=plaque forming unit(utilized for cell infection. It should be emphasized again that such aneffect on RNA synthesis may provide the key for the activity of thecompound on the virus and account for the action on the cells.

Assays using influenza virus

The plaque bioassays described above were carried out with influenzavirus. A fraction consisting of a mixture of (1) and (2) was tested foractivity on influenza virus A/Port-Chalmers/73. The results arecollected in Table 1.

It can be seen that 96% of inhibition is obtained with a concentrationas low as 0.15 μg/ml, when all concentrations above it show virusinhibition of 100%. The cells in the higher concentrations looked normalthroughout the experiments. In a control experiment, similarconcentrations of the same mixture were left in contact with the tissuecultures alone (uninfected) and they developed normally to produce thecells monolayers.

                  TABLE 1                                                         ______________________________________                                        In vitro plaque assay for a mixture of (1) and (2) with influenza             ______________________________________                                        virus                                                                         Concentrations                                                                             1.5    0.75   0.37 0.19 0.09 Control                             in ug/ml                                                                      Number of plaques                                                                          0      0      0    8    42   200                                 Inhibition % 100    100    100  96   79                                       ______________________________________                                    

Assays with Herpes simplex virus, strain KOS (HSV-1)

In these experiments the concentration of the stock solutions wasdetermined using a calibrated concentration curve prepared withultraviolet light at 280 mm. The saturated solutions of the respectivetwo compounds were filtered and the transmittance plotted on the curvefor the determination of the concentration. Higher accuracy was therebyreached.

The reslts are given in Table 2 for the different dilutions. They showthat hypericin was active at a concentration of 1.5 ng/ml (10⁻⁹ gr) andpseudo-hypericin at a concentration of 472 ng/ml.

                  TABLE 2                                                         ______________________________________                                        Anti-viral activity of hypericin and                                          pseudo-hypericin on Herpes simplex HSV-1                                                     No. of plaques                                                                          Inhibition                                                          per plate (%)                                                  ______________________________________                                        Final concentration                                                           of hypericin                                                                  250 ng/ml (10.sup.-9 gr)                                                                       0           100                                              25 ng/ml         0           100                                              2.5 ng/ml        0           100                                              0.25 ng/ml       1176        9                                                0                1288                                                         Final concentration                                                           of pseudohypericin                                                            4720 ng/ml (10.sup.-9 gr)                                                                      0           100                                              472 ng/ml        1           100                                              47.2 ng/ml       1488        (-15)                                            4.72 ng/ml       1000        0                                                0                1288                                                         ______________________________________                                    

In view of the above it is apparent that compositions according to theinvention are effective when they contain small amounts of the activeingredient. These are generally in the range of about 5 to 10 mg/kg ofbody weight.

Various forms of administration can be resorted to. For afflictions ofthe skin or of mucous membranes, they are advantageously used in topicalforms such as lotions or ointments, for example in polyethylene glycol.These will typically contain from about 1 to 5% hypericin or from about2 to 5% pseudohypericin weight per volume. The active principles, i.e.,hypericin and pseudohypericin, may also be administered in combinationin any desired ratio.

In addition to topical administration, any form of systemicadministration can be used. The dosage of active principle should besuch that a sufficient amount is present in the system to permiteffectiveness against viruses upon contact therewith wherever they maybe found in the system. While a certain amount of empiricalexperimentation will be necessary to determine appropriate dosagesdepending on the mode of administration (oral, parenteral, aerosol,etc.) and the particular virus being treated, no undue experimentationis required by one of ordinary skill in this art aware of thepharmacological activity of the active principles discussed herein.Determination of the effective amounts is within the skill in the art.

The present invention further includes salts or other derivatives ofhypericin or pseudohypericin which retain their anti-viral activity.Salts in which the base is of the alkaline or amine type areparticularly comprehended within the scope of the present invention.

In addition to the hypericin or pseudohypericin, the pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Preferably, the preparations, particularly those for oraladministration, such as tablets, dragees and capsules, and alsopreparations which can be administered rectally, such as suppositories,as well as suitable solutions for administration by injection or orally,or for administration by means of an aerosol contain from about 0.1 to99%, preferably from about 25-85% of active compound(s), together withthe excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating or dragee-making processes. Thus,pharmaceutical preparations for oral use can be obtained by combiningthe active compound(s) with solid excipients, optionally grinding aresulting mixture and processing the mixture of granules, after addingsuitable auxiliaries, if desired or necessary, to obtain tablets ordragee cores.

Suitable excipients are, in particular, fillers such as sugars, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or algenic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, steric acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions may beused, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize different combinations ofactive compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin and a plasticizer such as glycerol orsorbitol. The push-fit capsules can contain the active compounds in theform of granules which may be mixed with fillers such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In particular, it is alsopossible to use gelatin rectal capsules which consist of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleatetriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension including, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers.

A preferred form of administration may be by aerosol spray, either as ameans of systemic administration or as a means of topical administrationto the mucous membranes and lungs. Any conventional aerosol excipientsand propellants may be used for this purpose. For aerosoladministration, the active principles in accordance with the presentinvention may be packaged in a pressurized container with an appropriatesystem of valves and actuators. Preferably, metered valves are used withthe valve chamber being recharged between each actuation or dose, all asis well known in the art.

While the present description refers to the treatment of humans, theinvention includes the treatment of all animals having viral infections.Foremost among such animals are humans; however, the invention is notintended to be so limiting. It is within the contemplation of theinvention to treat any and all animals which may experience thebeneficial effects of the invention.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

LEGEND TO FIGS.

FIG. 1 Delay of destruction of VSV infected cells in presence ofdifferent concentrations of hypericin (1).

FIG. 2: Delay of destruction of uninfected cells in presence ofdifferent concentrations of hypericin (1).

FIG. 3: Incorporation of ³ H-uridine during RNA synthesis by: ΔVSV (50λ/ml) infected cells, and □ uninfected cells; measurements are made atdifferent concentrations of pseudohypericin (2).

FIG. 4: Incorporation of ³ H-uridine during RNA synthesis by: ΔVSV (100λ/ml) infected cells, and □ uninfected cells; measurements are made atdifferent concentrations of pseudohypericin (2).

We claim:
 1. A topical antiviral pharmaceutical composition comprising,a pharmaceutically acceptable excipient suitable to place thecomposition into form for topical administration and, as activeingredient, an antiviral effective quantity of a pure compound selectedfrom the group consisting of hypericin, pseudohypericin, apharmaceutically acceptable salt of hypericin or pseudohypericin, andmixtures thereof in any desired ratio.
 2. A composition in accordancewith claim 1, wherein said active ingredient is hypericin or aphrmaceutically acceptable salt thereof.
 3. A composition in accordancewith claim 1, wherein said active ingredient is pseudohypericin or apharmaceutically acceptable salt thereof.
 4. A composition in accordancewith claim 1, wherein said active ingredient is a combination ofhypericin, or a pharmaceutically acceptable salt thereof, andpseudohypericin, or a pharmaceutically acceptable salt thereof.
 5. Acomposition in accordance with claim 1, wherein said excipient is alotion or an ointment.
 6. A compound in accordance with claim 1, whereinsaid excipient includes polyethylene glycol.
 7. A composition inaccordance with claim 1, containing from about 5-10 mg per unit dosageof said active ingredient.
 8. A composition in accordance with claim 1,containing from about 1 to about 5% of said active ingredient.
 9. Amethod of treating vial infections in a mammal afflicted with a viralinfection, comprising administering to said mammal an effectiveantiviral amount of a substantially pure compound selected from thegroup consisting of hypericin, pseudohypericin, a pharmaceuticallyacceptable salt of hypericin or pseudohypericin, and mixtures thereof inany desired ratio.
 10. A method in accordance with claim 9, wherein saidadministering step comprises topically administering said compound to anarea infected with a virus.
 11. A method in accordance with claim 9,wherein said administering step comprises parenterally administeringsaid compound.
 12. A method in accordance with claim 9, wherein saidcompound is hypericin or a pharmaceutically acceptable salt thereof. 13.A method in accordance with claim 9, wherein said compound ispseudohypericin or a pharmaceutically acceptable salt thereof.
 14. Amethod in accordance with claim 9, wherein said compound is acombination of hypericin, or a pharmaceutically acceptable salt thereof,and pseudohypericin, or a pharmaceutically acceptable salt thereof. 15.A method in accordance with claim 9, wherein said compound isadministered in an amount of 5-10 mg per kg of body weight.
 16. A methodin accordance with claim 9, wherein said mammal is afflicted with avirus selected from the group consisting of vesicular stomatitis,influenza, herpes simplex, HSV-1 and HSV-2.